The objective of the proposed research is to characterize the mechanisms of Ca2+ entry and egress in pyramidal and nonpyramidal neurons of the rat neocortex. The dynamics of this process are crucial in determining the firing characteristics of neurons, synaptic plasticity and activation of metabolic pathways. Part of the objective of the project is to determine if the differences in the firing characteristics of pyramidal and nonpyramidal cells are the result of different rates of Ca2+ influx and mechanisms for buffering Ca2+, and their impact upon Ca2+-activated-K+ channels. The specific objectives of the proposed research are: 1. To identify and characterize the channels that allow Ca2+ influx through the plasma membrane. 2. To measure the rate of Ca2+ buffering in the cytoplasm of pyramidal and nonpyramidal cells and to explore the influence of the Ca2+-binding proteins, parvalbumin and calbindin, on the kinetics of this process. 3. To identify the active processes involved in the extrusion of Ca2+ from the cytoplasm. 4. To identify intracellular organelles responsible for Ca2+ release and the pathways for their activation. Whole-cell voltage-clamp experiments will be performed on acutely dissociated neurons from the mature rat visual and somatosensory cortices, to identify channels allowing Ca2+ entry and pumps actively removing Ca2+ from the cytoplasm. In the same cells the Ca2+-sensitive probe fura-2 will be used to monitor the kinetics of intracellular Ca2+ following various perturbations. After the physiological experiments immunocytochemical techniques will be used to test for the presence of parvalbumin or calbindin. The long term objective of the project is to understand the impact of intracellular Ca2+ regulation on the firing characteristics of neurons, that fundamentally shape the computational capabilities of the neocortex. Malfunctions in Ca2+ regulation have been implicated in many diseases (eg. Alzheimers and epilepsy). A detailed understanding of all the stages involved in Ca2+ regulation should allow the development of specific pharmacological interventions to ameliorate nervous disorders.